The V. cholerae regulator VpsR shares overall homology with Enhancer Binding Proteins that activate RNA polymerase containing the alternate sigma factor sigma54, but it lacks the conserved residues needed to bind to this polymerase and to hydrolyze ATP. In addition, transcription from the VpsR-activated promoter for genes of biofilm biogenesis, PvpsL, does not require sigma54 in vivo. We have developed the first in vitro transcription system using RNA polymerase containing the primary sigma factor (sigma70), VpsR, and PvpsL. We have demonstrated that RNA polymerase containing sigma70 is activated at PvpsL and that both VpsR and c-di-GMP are required for this activation. Using this system, we are determining the step(s) in transcription initiation that requires VpsR/c-di-GMP for activation and determining the protein-protein and protein-DNA interactions needed for the activated complex. Our overall goal is to construct a molecular map of VpsR/c-di-GMP/RNA polymease/DNA in the active transcription complex, and to investigate the role of c-di-GMP in VpsR structure and function. Our studies have shown that the presence of c-di-GMP does not significantly change the affinity of VpsR for PvpsL DNA or affect the ability of VpsR to dimerize. However, DNaseI footprints reveal that the activated PvpsL/VpsR/c-di-GMP/sigma70 RNA polymerase complex adopts different protein-DNA interactions from those formed by PvpsL/sigma70 RNA polymerase with or without c-di-GMP or VpsR. Our results are consistent with the idea that c-di-GMP is needed to generate a conformation that can promote the formation of the activated transcription complex and suggest a new paradigm in c-di-GMP-dependent transcription activation.